Recombinant protein delivery enables modulation of the phototransduction cascade in mouse retina

Inherited retinal dystrophies are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca2+. Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein 1 (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). The analysis of protein biodistribution and the direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful complementary tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-023-05022-0.


Figures S1 to S9
Table S1 Captions for Movies S1 to S5 Other Supplementary Materials for this manuscript include the following:

Figure S1
Assessment of the conjugation of GCAP1 with CF640R and removal of free dye.a) 15% SDS-PAGE of GCAP1 before and after conjugation with CF640R and after encapsulation in LPs.Lanes: M) marker, 1) LP-GCAP1 CF640R , 2) free-GCAP1 CF640R , 3) free-CF640R, 4) WT-GCAP1, stained with Coomassie blue (top panel) and upon excitation at 580 nm (bottom panel).b) Representative absorption spectra of the flowthrough of the 4 sequential washing steps to remove unconjugated dye.

Figure S2
Representative profiles of the size of a) 5.1 nM LP-empty (black), b) 4.3 nM LP-E111V-GCAP1 (red), c) 4.6 nM LP-GCAP1 CF640R (blue) estimated by NTA.Monitoring of the size of ~2.9 nM (Table S1) LP-E111V-GCAP1 after d) 1 (green), e) 90 (yellow) and f) 180 days (violet).Each plot represents the average of 3 independent measurements, standard errors are displayed as a lighter shade of the color of each trace.Concentrations refer to the stock solutions, before dilutions required for NTA analysis.g) LP-CF640R appear as point-like (diffraction limited) fluorescence when suspended in agarose gel (left field) while empty LPs do not (right field).The two fields were acquired, processed, and displayed with identical parameters.Scale bar 10 µm.

Figure S3
Representative images of cGFP cell line after 24 h incubation with 100 µl of 104 µM free-

Figure S6
Representative central plane of Z-stack of retinal cryosections after 24 h incubation with 180 µl of 99.6 µM free-GCAP1 His (left panel) and 4.5 nM LP-GCAP1 His (right panel).Sections were stained with an anti-His antibody (red) using the same image acquisition and display parameters (compare with Fig. 4 in the main text, where nuclei have been stained).Note the presence of more diffused and speckled signals in the case of delivered free-protein, and the prevalence of diffused signal in the case of LP-encapsulated protein at this time frame.

Figure S8
Example of the effect of free-E111V-GCAP1 in slowing scotopic flash response kinetics.BaCl2 (50 µM) was first injected in the 2 ml well followed, after 45 min, by injection of 100 µl free-E111V-GCAP1.The control flash family (blue traces) was recorded just before protein injection, while the treated one (purple traces) after 37 min.Each trace is the average of several responses and the data shown here is from one of the experiments that also contributed to fig.6h.Note that the effect of the free protein on response kinetics is underestimated in this figure, since the untreated retina in this pair showed a slight acceleration of kinetics over the same period.This highlights the importance of analyzing the effect of the injected formulations by normalizing 'treated' over 'control' retinas, as done in Fig. 6 (see also the Methods section).

Movie S1
The three-dimensional structure of GCAP1 is shown as light-grey cartoon with the molecular surface in transparency, Ca 2+ -ions are displayed as green spheres, the sidechains of Lys residues are labelled represented as red sticks with N atoms in blue.The molecular surface of the primary amines belonging to Lys sidechains is shown in blue in transparency.

Movie S2
Live-cell imaging at 6 h of cGFP cell line incubated with 100 µl of 140 µM free-CF640R, snapshots were acquired at a 30 min interval, green fluorescence refers to eGFP, red fluorescence refers to free-CF640R molecules.

Movie S3
Live-cell imaging at 6 h of mGFP cell line incubated with 100 µl of 104 µM free-GCAP1 CF640R , snapshots were acquired at a 30 min interval, green fluorescence refers to eGFP, red fluorescence refers to free-GCAP1 CF640R molecules.

Movie S4
Live-cell imaging at 6 h of cGFP cell line incubated with 100 µl of 104 µM free-GCAP1 CF640R , snapshots were acquired at a 30 min interval, green fluorescence refers to eGFP, red fluorescence refers to free-GCAP1 CF640R molecules.

Movie S5
Live-cell imaging at 24 h of mGFP cell line incubated with 100 µl of 4.3 nM LP-GCAP1 CF640R (containing in the aqueous core the equivalent number of GCAP1 CF640R molecules present in a 104 µM solution) snapshots were acquired at a 30 min interval, green fluorescence refers to eGFP, red fluorescence refers to LP-GCAP1 CF640R .

GCAP1
CF640R after replacing cell medium with FluoroBrite DMEM.Left panel shows eGFP fluorescence, center panel shows CF640R fluorescence, left panel shows merged fluorescence.

Figure S9 Method
Figure S9